Maria Cristina Bronharo Tognim

Emergence of an IMP-like metallo-enzyme in an Acinetobacter baumannii clinical strain from a Brazilian teaching hospital

Multidrug-resistant Acinetobacter spp. constitute a serious cause of nosocomial infection in Brazilian hospitals. This manuscript reports the first appearance of an IMP-like metallo-beta-lactamase encountered in a clinical isolate of A. baumannii from a Brazilian teaching hospital.

Polymyxin-Resistant Acinetobacter spp. Isolates: What is Next?

To the Editor: In Brazilian hospitals, Acinetobacter spp. has been an important etiologic agent of nosocomial infections, mainly pneumonia (1–3). In general, ampicillin/sulbactam and carbapenems remain the last therapeutic options for treatment of such infections (3,4). However, resistance rates to carbapenems have increased, reaching rates approximately 12% or higher in some Brazilian hospitals (1,3,4). Thus, more toxic agents such as polymyxins have been used as alternative therapeutic drugs against multidrug-resistant Acinetobacter infections (5,6). The clinical use of polymyxins has been based on antimicrobial susceptibility results and previous clinical experience. However, the National Committee for Clinical Laboratory Standards (NCCLS) documents do not currently provide interpretative criteria for the testing of polymyxins (7). In addition, the disk diffusion technique was reported to be an unreliable method for evaluating the susceptibility to polymyxins (8). Since Acinetobacter clinical specimens exhibiting high MICs for polymyxins (MIC, 8–32 μg/mL) were recently detected, we searched for the frequency of occurrence of Acinetobacter spp. strains exhibiting reduced susceptibility to polymyxin B among 100 bloodstream isolates of Acinetobacter spp (8). The bacterial isolates were consecutively collected between September 1999 and December 2000 from a tertiary Brazilian hospital, where Acinetobacter spp. infections have reached endemic levels and polymyxins have been frequently used. Only one isolate per patient was included in the study. The isolates were identified to the species level using the BBL Crystal System (Becton Dickinson, Sparks, MD). The susceptibility to polymyxin B and meropenem were tested by disk diffusion and agar dilution techniques according to NCCLS recommendations (9,10). The susceptibility interpretative criteria for meropenem and polymyxin B were based on the current and former NCCLS documents, respectively (7,11). The MIC was defined as the lowest antimicrobial concentration that inhibited bacterial growth. Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922 were used as quality control strains. Testing errors and agreements were determined by comparing the results of the disk diffusion with the standard criterion agar dilution method. Categorical agreement was obtained when the isolates were classified within the same susceptibility category. The very major and major errors were related to false susceptibility and false resistance results, respectively. To evaluate whether the polymyxin B-resistant strains isolates were epidemiologically related, these isolates were molecularly typed by pulsed-field gel electrophoresis (PFGE) as previously described (12). PFGE patterns were considered identical if they shared every band, similar if they differed from one to three bands, and distinct if they differed by four or more bands (12). Despite the limitation of commercial systems for identifying the genus Acinetobacter at species level, Acinetobacter baumannii (80.0%) was the most commonly identified species, followed by A. lwoffi (4.0%). Sixteen percent of the Acinetobacter isolates were not identified to species level by the BBL Crystal System. Meropenem (MIC50, 1 μg/mL) and polymyxin B (MIC50, 1 μg/mL) showed similar in vitro potency. However, meropenem exhibited the highest susceptibility rate (99.0% susceptible). In contrast to previous studies, only one strain was resistant to meropenem (1–3,8), which indicates that the carbapenem-susceptibility rates among Acinetobacter spp. isolates may vary according to the period evaluated even in the same institution. By using the polymyxin B resistance breakpoint (MIC >4 μg/mL) presented by the former NCCLS document, which was recently validated, we found that five Acinetobacter spp. isolates were considered resistant to polymyxin B (MICs, 8–32 μg/mL) (8,11). All isolates were susceptible to meropenem and belonged to A. baumannnii (4) and A. lwoffi (1) species. The polymyxin B–resistant isolates were categorized as susceptible by disk diffusion (100%, very major error). The disk diffusion method is widely used in Brazil and worldwide. However, disk diffusion was confirmed to be an unreliable test for detecting Acinetobacter spp. isolates with reduced susceptibility to polymyxins. These results are in agreement with those previously reported (8). Among the five polymyxin B–resistant Acinetobacter spp., four distinct patterns were characterized by PFGE. Two polymyxin B–resistant strains, which were isolated from different units of the Sao Paulo Hospital complex, shared an identical PFGE pattern. The PFGE results suggest that the polymyxin B use may have played a role in the selection of resistant strains. On the other hand, two isolates shared an identical PFGE pattern, which raises the possibility of patient-to-patient transmission of epidemic strains. Intra- and interhospital dissemination of multidrug-resistant Acinetobacter spp. clones has already been reported in Brazilian hospitals (13). Our findings suggest that the polymyxin B–resistant strains have emerged because of antimicrobial selective pressure and dissemination of clonal strains. Further epidemiologic studies are necessary to correlate the emergence of polymyxin-resistant Acinetobacter spp. isolates to the clinical response with polymyxin B therapy. Since the emergence of polymyxin B resistance may leave no efficacious drugs for the treatment of infections caused by multidrug-resistant Acinetobacter spp. isolates, strict infection control measures must be adopted to avoid the emergence and spread of such isolates. The low accuracy of routine susceptibility tests, especially disk diffusion, may jeopardize rapid implementation of such measures.

Dissemination of IMP-1 Metallo-β-Lactamase–Producing Acinetobacter Species in a Brazilian Teaching Hospital

OBJECTIVE To evaluate the emergence and dissemination of metallo- beta -lactamase (MBL)-producing Acinetobacter species. DESIGN All carbapenem-resistant Acinetobacter strains (1 strain per patient) collected during the period 1993-2001 were evaluated. SETTING A Brazilian tertiary care teaching hospital (Hospital Sao Paulo, Sao Paulo). METHODS Seventy-three strains of carbapenem-resistant Acinetobacter species were recovered from the organism bank of the hospital. All isolates were tested for antimicrobial susceptibility by broth microdilution methods, and the production of MBL was initially assessed by phenotypic tests (MBL Etest strip and a disk approximation test). The MBL enzymes were identified by polymerase chain reaction using primers for bla(IMP), bla(VIM), and bla(SPM), followed by gene sequencing. Genetic similarity among the carbapenem-resistant strains was evaluated by automated ribotyping. RESULTS Only colistin and ampicillin-sulbactam showed reasonable in vitro activity against carbapenem-resistant isolates (97% and 74% of isolates susceptible, respectively). More than half of the isolates (55%) had a positive MBL phenotypic test result and a positive polymerase chain reaction result for bla(IMP-1). The proportion of IMP-1-producing Acinetobacter isolates among carbapenem-resistant strains increased from 0% in the 1993-1997 period to 29% in 1998 and 100% in the 1999-2001 period. No carbapenem-resistant Acinetobacter isolates that harbored bla(VIM) or bla(SPM) were detected. Molecular typing results revealed 20 ribogroups among carbapenem-resistant isolates. During the study period of 1994-2001, we identified 2 major ribogroups, 52-1 (MBL-negative and MBL-positive strains) and 60-7 (MBL-positive strains), that had a coefficient of similarity of 0.85 or higher. CONCLUSIONS Our results indicate that IMP-1-producing strains of Acinetobacter emerged in our institution in 1998. Since then, production of this MBL was detected not only in the major ribogroups of carbapenem-resistant Acinetobacter species but also among isolates that belonged to 17 distinct ribogroups, indicating that this important mechanism of antimicrobial resistance was disseminated among distinct clones.

Endocarditis due to glycopeptide-intermediate Staphylococcus aureus: case report and strain characterization

We report a case of infective endocarditis due to vancomycin-intermediate Staphylococcus aureus (VISA) that did not respond to high doses of vancomycin. Initial vancomycin MIC of the last isolate recovered from blood was 8 micro g/mL, but could be induced up to 32 micro g/mL by consecutive growing with vancomycin. Clinical response was only accomplished when linezolid was included in therapy.

Outbreak of Klebsiella pneumoniae carbapenemase–producing K pneumoniae: A systematic review

BACKGROUND First detected in the United States in 1996, Klebsiella pneumoniae carbapenemase (KPC) has spread internationally among gram-negative bacteria, especially K pneumoniae. These microorganisms can cause serious infections in hospitalized patients, and there are few therapeutic options, culminating in increased mortality. The objective of this study was to describe the occurrence of outbreaks that were caused by KPC-producing K pneumoniae, emphasizing the interventions that were implemented to contain the outbreaks. METHODS PubMed, Web of Knowledge, and Literatura Latino Americana em Ciências da Saúde databases were searched for articles that were published between 2001 and 2012 according to the recommendations of Preferred Reporting Items for Systematic Reviews and Meta-Analyses. RESULTS Of the 586 studies identified, 13 were selected for the final sample. Most studies showed that the containment of KPC outbreaks is possible in hospital settings through several actions, particularly use of surveillance cultures and the establishment of contact precautions. CONCLUSIONS The results show that limiting the cross-transmission of these and other KPC-producing bacteria is possible in a hospital setting. However, such isolates need to be detected early with the aid of culture surveillance and contained early using appropriate actions immediately to prevent an outbreak.

Preliminary evaluation of adherence on abiotic and cellular surfaces of Acinetobacter baumannii strains isolated from catheter tips

The cell surface hydrophobicity and adhesion to abiotic and cellular surfaces was tested in five clinical strains of Acinetobacter baumannii isolated from catheter tips. Biochemical and molecular characteristics of these strains were also studied. Hydrophobicity was characterized by a test for affinity to xylene. Adhesion to abiotic surfaces (polystyrene, formica, latex and glass) was evaluated in Petri plates using the stamp technique. Buccal epithelial cells were used for tests of adhesion to cellular surfaces. Adhesion to the catheter was evaluated by repeatedly rinsing the catheters and rolling them over nutrient agar. Molecular typing of the strains was done by the ERIC-PCR technique. The degree of hydrophobicity of the strains varied from hydrophobic to hydrophilic. All the strains adhered to the cell surfaces and to the catheters, and three of them strongly adhered to latex, polystyrene and formica. Catheter adhesion was reduced by meropenem. We found a direct relationship between the degree of bacterial hydrophobicity and adhesion to the abiotic surfaces, but not with adhesion to cellular surfaces, which suggests that different mechanisms are involved in adherence.

Quinolone-resistant Escherichia coli

Quinolones (nalidixic acid--NAL, norfloxacin--NOR, ciprofloxacin--CIP and gatifloxacin--GAT) were tested against Escherichia coli isolated from urine (385 patient samples) by disk diffusion (DD) and agar dilution (AD) methods. Fifty-three samples (13.8%) were classified as resistant to at least one of the quinolones tested. CIP and NOR susceptibilities were the same (91.4%) and they were similar to GAT (92.7%). Susceptibility to NAL, detected by the disk diffusion method, was used to predict susceptibility to NOR, CIP and GAT by the agar dilution method. The sensitivity and specificity of NAL were 100% and 95%, respectively. Twelve samples were analyzed for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes. Sequencing of these genes failed to find any mutations in the quinolone-sensitive isolates. However, three mutations were observed in the isolates resistant to all the quinolones tested--two in gyrA and one in parC. A single mutation in gyrA was found in the strains that were resistant to nalidixic acid but fluoroquinolone-sensitive. These findings support the suggestion that NAL could be used as a marker for susceptibility to fluoroquinolones in routine microbiology laboratories. The overall resistance rate to quinolones in the present study was 13.8%, which is higher than that observed in other studies carried out in developed countries. Our findings serve as a warning that resistance to this group of antimicrobial agents is increasing.

Prevalence and Genetic Characterization of bla CTX-M Among Klebsiella pneumoniae Isolates Collected in an Intensive Care Unit in Brazil

Abstract Thirteen (44.8%) CTX-M-2-producing K. pneumoniae clinical isolates were identified among 29 strains collected from single patients with serious infection hospitalized in an intensive care unit of a tertiary hospital located in São Paulo, Brazil. These isolates belonged to 9 different typing clusters and showed great diversity of plasmid content. Their bla CTX-M-2 was carried in an ISCR1/sul1-type integron structure located in transferable plasmids of different sizes or in the chromosome.

Avaliação da acurácia de testes laboratoriais para detecção de amostras de Klebsiella pneumoniae produtora de betalactamase de espectro estendido

Bacterial strains producing extended spectrum beta-lactamases (ESBL) represent a common resistance problem among Brazilian hospitals. Due to the difficulty of ESBL detection in the clinical laboratory, these bacterial isolates require a reproducible, efficient, and low cost detection method. The aim of the present study was to evaluate the efficacy of detection of K. pneumoniae ESBL isolates by two methods: the Etest ESBL strip and the inhibitor potentiated disk diffusion test with clavulanic acid (clavulanate-potentiation test). The sensitivity and the specificity of beta-lactam agents against these isolates were also evaluated. The experiments were performed on a total of 134 K. pneumoniae isolates recovered from blood specimens in our institution from July 1996 to July 2001. The samples were tested for ESBL production by the NCCLS screen test, clavulanate-potentiation test and Etest ESBL strip. Isolates presenting positive results for the screen test and for at least one of the evaluated tests were considered ESBL producers (gold standard). The results of this study yielded a 100% specificity and sensitivity for the clavulanate-potentiation test, and the best indicators of ESBL production were cefotaxime and cefpodoxime. The Etest ESBL strip also turned out to be a very sensitive (96%) and specific (100%) method, being cefotaxime the most efficient substrate. According to the results of this investigation, the clavulanate potentiation disk diffusion test displayed an excellent performance and can be easily implemented in routine clinical laboratories as a practical, reliable, and accurate method.

Epidemiologic surveillance of multidrug-resistant bacteria in a teaching hospital: A 3-year experience

Background: The objective of this prospective study was to verify the effectiveness of a multidisciplinary surveillance program that was implemented in a teaching hospital in southern Brazil, to prevent and control the spread of multidrug‐resistant organisms. Methods: The program implemented involved establishment of prevention guidelines, hand‐hygiene promotion, isolation of patients colonized or infected by such organisms, enforced contact precautions, and terminal cleaning and disinfection of isolation rooms. A microbiology service, previously provided by an external laboratory, was established in the hospital. Detection of bacteria‐resistant genes and molecular typing were performed also. Results: Statistically significant differences were observed between the pre‐ and post‐intervention periods (P = .00198). Control measures were effective in blocking the dissemination of a previously endemic clone of Acinetobacter baumannii. Changes were observed in the dissemination pattern, from a monoclonal to a polyclonal mode. The incidence of vancomycin‐resistant Enterococcus during the surveillance period was low. Only 2 isolates of BLAKPC‐positive Klebsiella pneumoniae (distinct profiles), and 5 isolates of BLASPM‐positive Pseudomonas aeruginosa (a single cluster), were detected. Conclusions: These results indicate that the surveillance program implemented was effective in preventing the spread of multidrug‐resistant organisms in the hospital.